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Applications of BBa_K649303
We confirmed that E. coli,introduced ispS, produced isoprene, by means of using Gas Chromatography-Mass Spectrometry equipment.
1. PlacIQ on pSB3K3
2. PlacIQ-RBS-ispS on pSB3K3
Each bacterial sample was grown in a 500 mL flask containing 100 mL LB media. Cultures were grown at 37℃ and then induced by 0.5 mM IPTG when OD600 reached 0.6. After 4 hours of induction, 50 mL of headspace gas was taken by absorbing material (mini-PAT including Tenax: Japan Analytical Industry Co., Ltd) and injected into GC-MS.
We calculated the amount of isoprene by calibration date we obtained. X represents the area and Y represents the amount of isoprene [mg]. The calibration curve is described by the equation,
Y = 10-7.9 × X0.89.
According to the calibration curve, we detected 4.1×10-5 mg/L isoprene produced by E. coli BL21 (DE3) introduced ispS, while negative control (PlacIQ) produced one eighth of our new E. coli.
[Mechanism of GC-MS]
To measure the amount of isoprene produced by our E. coli, we used electron-ionization Gas Chromatography-Mass Spectrometry equipment (GC-MS, QP-2010, SHIMADZU, Japan). Analytes were separated by a nonpolar column (Rtx-1MS: Length 30 m, ID 0.25 mm film thickness 0.5 µm, USA) working in a constant flow mode (2.99 mL min-1). The temperature program was chosen as follows: 40℃ for 7 min, increase to 280℃ at rate of 10℃ min-1, 280℃ for 5 min. The mass spectrometer worked in SIM mode, m/z 67. The retention time of isoprene is very short (about 1.06-1.10 min).
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